Food and/or feed compositions for preventing and treating inflammatory diseases

ABSTRACT

This invention relates generally to compositions and methods for combating inflammatory disease and particularly to the use of food and/or feed compositions for preventing, reducing and/or treating inflammatory disorders, diseases, or discomforts, wherein the compositions comprising at least one specific pectin.

This application is a divisional of commonly owned U.S. application Ser.No. 15/741,584, filed Jan. 3, 2018 which is the U.S. national phase ofInternational Application No. PCT/EP2016/066222 filed Jul. 8, 2016,which designated the U.S. and claims priority to EP Patent ApplicationNo. 15176190.5, filed Jul. 10, 2015, the entire contents of each ofwhich are hereby incorporated by reference.

JOINT RESEARCH AGREEMENT

The claimed invention was made by, on behalf of, and/or in connectionwith one or more of the following parties to a jointuniversity-corporation research agreement: Wageningen UniversityDepartment Agrotechnology and Food Sciences, Universitair MedischCentrum Groningen Department Pathology and Medical Biology,Rijksuniversiteit Groningen Department Neuroendorinology, DSM FoodSpecialties, Nutrition Sciences N.V., and Agrifirm Innovation Center,which is legally part of CCL B.V., a subsidiary of Agrifirm Group. Theagreement was in effect on and before the date the claimed invention wasmade, and the claimed invention was made as a result of activitiesundertaken within the scope of the agreement.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing (Name:4662_3898_ST25.txt; Size: 789 bytes; and Date of Creation: Mar. 6, 2020)is incorporated herein by reference in its entirety.

This invention relates generally to compositions and methods forcombating inflammatory diseases and particularly to the use of foodand/or feed compositions for preventing, reducing and/or treatinginflammatory disorders, diseases, or discomforts.

Inflammatory diseases (or also inflammation-related disorders) in humanare for example diseases such as Alzheimer's, ankylosing spondylitis,arthritis (osteoarthritis, rheumatoid arthritis (RA), psoriaticarthritis), asthma, atherosclerosis, Crohn's disease, colitis,dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowelsyndrome (IBS), systemic lupus erythematous (SLE), nephritism,Parkinson's disease, ulcerative colitis, auto-immune disease, asthma,atopy, cardio-vascular disease, diabetes, immune senescence,ischemia/reperfusion injury of the heart or of kidneys, felineinfectious peritonitis, mastitis, psoriasis, sepsis, systemic lupuserymathosis, tumour metastasis, and visceral or cutaneous Leishmaniasis.Also it can be used to prevent or cure pro-inflammatory events such asoccur during the use of cytostatics for cancer treatment, or otherdiscomfort during the use of drugs that causes visceral damage.

Most of these Diseases are Wide-Spread.

For example Arthritis is the most common cause of disability in the USA.More than 20 million individuals with arthritis have severe limitationsin function on a daily basis. It can affect humans as well as animals.In the context of the present invention we use the term “patients” todescribe the targeted individuals. These patients are susceptible to orsuffering from inflammatory disease. Rheumatism or rheumatic disorder isa non-specific term for medical problems affecting the joints and/orconnective tissue. The term “rheumatism” is still used in colloquialspeech and historical contexts, but is no longer frequently used inmedical or technical literature; there is no longer any recognizeddisorder simply called “rheumatism.” The traditional term covers such arange of different problems that to ascribe symptoms to “rheumatism” isnot to say very much. “Non-articular rheumatism,” also known as“regional pain syndrome” or “soft tissue rheumatism,” can causesignificant discomfort and difficulty. Furthermore, arthritis andrheumatism between them cover at least 200 different conditions

Rheumatism and arthritis are general terms for acute and chronicconditions characterized by inflammation and pain. Rheumatism is ageneral category of conditions characterized by inflammation and pain inmuscles and joints, including arthritis. Arthritis is characterized byinflammation of joints that causes swelling and pain. Types of arthritisinclude osteoarthritis, rheumatoid arthritis, ankylosing spondylitis(AS), and systemic lupus erythematosus (SLE). Rheumatic conditionsinclude infectious arthritis, rheumatoid arthritis, arthritis due torheumatic fever, arthritis due to trauma or degenerative joint disease,myositis, neurogenic arthropathy, bursitis, fibromyositis andhydroarthrosis. The cause of such diseases in not always fullyunderstood but may be the result of other degenerative diseases, trauma,or auto-immune diseases such as SLE. Inflammation also occurs as adefensive response to host invasion by foreign agents and mechanicaltrauma that results in an immune response, e.g., microbial agents suchas bacterial and viruses, toxins, alarm molecules, and neoplasia.

What these diseases and conditions, examples of inflammatory diseases,share in common is inflammation and the resulting pain. Prior methodsfor preventing and treating inflammatory diseases have generally focusedon pain-killing and anti-inflammatory drugs. Typical methods havefocused on oral medications such as steroidal cortisone derivatives andnumerous non-steroidal anti-inflammatory drugs (NSAIDs). Unfortunately,these drugs almost always exhibit undesirable side effects. Otherefforts have focused on joint implants such as the knee or hip implants.These methods are lengthy and complicated surgical procedures that forcethe patient to undergo costly invasive surgery and a significantrecovery period requiring a rigorous and costly regimen of physicaltherapy. There is, therefore, a need for new methods for preventing andtreating inflammatory diseases that avoids the undesirable side effectsand costly surgical procedures characteristic of previous methods forpreventing and treating inflammatory diseases.

Another example in which the invention is applicable, is in managing orpreventing disease and/or discomforts and/or unwanted side-effectscaused by unhealthy diets or the use of drugs. For example mucositis isan inflammation in the small intestine caused by chemotherapy such asthe commonly applied doxorubicin. This drugs is causing mucosal damageand release of intracellular molecules such as DNA, RNA and heat shockproteins (also called alarm molecules or danger associated molecularpatterns) that cause a severe inflammatory response with mucositis as aconsequence. This can be prevented or reduced by this invention. This isconsidered to be, but not limited to, an example of inflammatoryresponses as a consequence of unhealthy diets, use of pharmaceuticals orthe use of drugs.

In animals gastrointestinal disturbances that involve inflammatoryresponses are widespread. In farm animals (pigs, poultry and ruminants)this phenomenon causes large economic losses. For example within the pigpopulation total losses of all piglets born in the European Union amountto approximately 17% and a substantial proportion of these losses can beassociated with infections via mucosal surfaces (Lallès et al., 2007,Proc Nutr Soc. 66(2):260-268). A transient anorexia in newly weaned pigsis considered a major cause of these problems that leads to gutdysfunction, increased sensitivity to enteric infections and diarrhea.Concomitant patho-physiological changes include 20-30% reduction inmucosal weight associated with villous atrophy (Lallès et al., 2004,Animal Research 53, 301-316), a compromised intestinal barrier function(Wijtten et al., 2011, Br J Nutr. 105(7):967-981), disturbances in thehomeostasis of the gut microbiota (Bauer et al., 2006, Nutr Res Rev.19(1):63-78) and in the anatomical and functional development of theimmune system (Lallès et al., 2007, Proc Nutr Soc. 66(2):260-268). Alsoin poultry, enteric diseases significantly contribute to economic lossesbecause of impaired animal performance, increased mortality, and reducedwelfare of birds (Timbermont et al., 2011, Avian Pathol. 40(4):341-347).Nonspecific enteritis, coccidiosis, viral infections, dysbacteriosis andbacterial infections have been identified as major intestinal diseasesthat cause wet litter in poultry (Hermans et al., 2006, Vet Rec.158(18):615-622). Although there are some species related differences,the underlying patho-physiological changes are quite similar to those inpigs. For instance, small intestinal development in newly hatchedbroilers deteriorates at low feed intake levels (Wijtten et al., 2012,Acta Agriculturae Scandinavica Section A-Animal Science, 62(1), 1-12).

In the past, in-feed antibiotic growth-promoting agents were usedprophylactically to prevent these kind of problems. Nowadays, theprophylactic use of several antibiotics in farm animals is banned in theEuropean Union or under increased pressure by the public opinion inother countries such as the United States due to concerns aboutantibiotic residues in food and increased antibiotic resistance inpathogens. Hence, also for animals there is a need for new methods forpreventing and treating gastrointestinal disturbances that involveinflammatory responses.

Surprisingly, it was found that a specific kind of pectin (in regard toits degree of esterification) is useful in preventing, reducing and/ortreating inflammatory disorders, diseases, or discomforts.

By “preventing, reducing and/or treating inflammatory disorders,diseases, or discomfort” it is mainly meant:

-   -   minimizing the risk of contracting any of the above listed        diseases, and/or    -   ameliorating the related symptoms, and/or    -   lessening pain or discomfort associated with any of the above        listed diseases and/or    -   lengthen the time between episodes where any of the above listed        diseases can occur and/or    -   improving human and animal overall health and welfare

Pectin is a structural heteropolysaccharide contained in the primarycell walls of terrestrial plants. It is produced commercially as a whiteto light brown powder, mainly extracted from citrus fruits, and is usedin food as a gelling agent, particularly in jams and jellies. It is alsoused in fillings, medicines, sweets, as a stabilizer in fruit juices andmilk drinks, and as a source of dietary fiber. Pears, apples, guavas,quince, plums, gooseberries, oranges, and other citrus fruits containlarge amounts of pectin, while soft fruits like cherries, grapes, andstrawberries contain small amounts of pectin. But also other plantsources than fruits can comprise pectin. For example, pectin can besourced from potato, soy, sugar beet, chicory, carrot, tomato, pea,parsnip, and (green) beans. All these lists are not exhaustive at all.Only the main sources are listed.

The following illustration shows parts of the structure of pectin aswell as (partially) esterified pectin.

Structure of Pectin (Polygalacturonic Acid)

Structure of (Partially) Methylated Pectin

In the above shown formula the esterification is a methylation, but alsoother groups can be used (such as acetyl). Pectin can be esterified withone group (such as CH₃ or COCH₃) or with more than one group in the sameoligomer structure. Acetylation usually occurs at the oxygen in thehydroxyl group on position 2 and/or 3, while methylation usually occursat the carboxyl group on position 5.

In the scope of the present invention the degree of esterification isused to describe the percentage of esterified pectin monomer units inthe backbone.

Pectin is a complex polysaccharide composed of a α-1,4-linkedD-galacturonic acid (GalA) backbone (the so-called homogalacturonan orsmooth region) and segments consisting of alternating sequences ofα-(1,2)-linked L-rhamnosyl and α-1,4-linked D-galacturonosyl residuesramified with side chains of arabinans, arabinogalactans and galactans(branched rhamnogalacturonans or hairy regions). Pectins are decoratedwith neutral sugars (NS), mainly being galactose and arabinose attachedto the rhamnose moiety in the backbone.

Commercial pectins usually contain low amounts of neutral sugar as aresult of the acid extraction (the neutral sugar content is around 5%).Other structural elements of pectins are xylogalacturonan andrhamnogalacturonan II. Rhamnogalacturonan II is carrying peculiar sugarresidues such as Api (D-apiose), AceA (3-C-carboxy-5-deoxy-L-xylose),Dha (2-keto-3-deoxy-D-lyxo-heptulosaric acid) and Kdo(2-keto-3-deoxy-D-manno-octulosonic acid). The relative proportions ofthese different structural elements may vary significantly for differentplant origins and the various derived commercial products.

The various structural elements of pectin can be esterified. The majortypes of esterification are: O-methyl, O-acetyl and O-feruloyl. Notexcluding any other types of esterification. Most of the esterificationsreside in the homogalacturonan region on the GalA residues. The GalAresidues can be thus present with free carboxyl groups or esterified atone or more of the carboxyl groups. Esterification can occur asmono-esterification, but also as double esterification of single GalAresidues. Not excluding any other numbers of esterification. Theesterification on a single residue can be through a single type of alkylgroup (i.e. methyl) or a single type of acyl group (i.e. acetyl). Notexcluding any mixed type esterification. Thus, GalA can be methylated(leading to 0 or 1 methyl groups per GalA residue) or can be acetylated(leading to 0, 1 or 2 acetyl groups respectively on the oxygen of thehydroxyl group on the C-2 and/or C-3). The latter occurs as such insugar beet and potato pectins.

The pectins of the current invention typically have a MW sizedistribution of 5 and 800 kDa. Preferably the MW size distribution isbetween 5 and 400 kDa. Preferably, the majority of the pectin moleculesin a given pectin composition has a MW below 200 kDa. Preferably, themajority of the pectin molecules in a given pectin composition has a MWbelow 100 kDa.

The degree of esterification (DE) is by definition the amount of esters(in moles) present per 100 moles of total galacturonic acids (free GalAand substituted GalA summed together). As most commercial pectins areessentially having esterifications of the methyl-ester type, the DE isoften expressed as the degree of methylation (i.e. DM). In that case,the degree of esterification is by definition the amount ofmethyl-esters (in moles) present per 100 moles of total galacturonicacids (free GalA and substituted GalA summed together). In the case thatthe esterification is of the acetyl type, the DE is often expressed asthe degree of acetylation (i.e. DA). In that case, the degree ofesterification is by definition the amount of acetyl-esters (in moles)present per 100 moles of total galacturonic acids (free GalA andsubstituted GalA summed together). In the case of multiple types ofesterification in a single pectin sample, the DE is often expressedsplitted in to a degree of methylation (i.e. DM) and a degree ofacetylation (i.e. DA). These are calculated as described above.Alternatively, the DE can be expressed as the degree of esterification,defined the by amount of galacturonic acid residues modified with one ormore esterifications—either being of the methyl or the acetyl type—(inmoles) present per 100 moles of total galacturonic acids (free GalA andsubstituted GalA summed together).

In the context of this invention the term degree of esterification (DE)is used, and the percentages described are always based on the amount ofGalA residues which are substituted through esterification (i.e.methylation). A DE of 50 means that 50% of all possible GalA residuesare esterified (i.e. methylated).

The following patent application is related to the use of esterifiedpectins, more specifically it is related to the use of esterifiedpectins with a specific degree of esterification.

The following distinction is made among the esterified pectins:

-   -   (i) Low-esterified pectins    -   (ii) High-esterified pectins.

Low-esterified pectins have a degree of esterification (DE) of less than50%. This means that less than 50% of the possible positions areesterified.

High-esterified pectins have a DE of more than 50%. This means that morethan 50% of the possible positions are esterified.

DEs values for commercial HM-pectins typically range from 60 to 75% andthose for LM-pectins range from 20 to 40% (Sriamornsak, 2003, SilpakornUniversity International Journal 3 (1-2), 206-228).

As mentioned above pectins are present in almost all higher plants.Several by-products of the food industries are used for theirextraction, such as citrus peels (by-product of citrus juiceproduction), apple pommace (by-product of apple juice manufacture),sugar beet (by-product of the beet-sugar industry) and in a minor extendpotatoes fibres, sunflower heads (by-product of oil production) andonions (May, 1990, Carbohydr. Polymers, 12: 79-99). A typical process toextract HM pectins from the pomace or peels is in hot diluted mineralacid at pH1-3 at 50-90° C. during 3-12 hours (Rolin, 2002, In: Pectinsand their Manipulation; Seymour G. B., Knox J. P., Blackwell PublishingLtd, 222-239). Dry citrus peels contain 20 to 30% of pectin on a drymatter basis, lower amounts are present in dried apple pomace (10 to15%) (Christensen, 1986, Pectins. Food Hydrocolloids, 3, 205-230). Byadding alcohol (usually isopropanol but methanol or ethanol are alsoused) the pectins are precipitated. Finally, the gelatinous mass ispressed, washed, dried and ground (May, 1990, Carbohydr. Polymers, 12:79-99). Depending on the process conditions, pectins with a DM from 55to 80% are obtained (Rolin, 2002, In: Pectins and their Manipulation;Seymour G. B., Knox J. P., Blackwell Publishing Ltd, 222-239).

Low-methylated (LM) pectins can be obtained by de-esterification ofhigh-methylated (HM) pectins mainly by controlling the acidity, thetemperature and the time during extraction. To produce other types ofpectins, esters can be hydrolysed by the action of acid or alkali eitherbefore or during an extraction, as concentrated liquid or in thealcoholic slurry before separation and drying. When alkali is used, thereaction has to be performed at a low temperature and in aqueoussolutions to avoid β-eliminative degradation of the polymers(Kravtchenko et al, 1992, Carbohydrate Polymers, 19, 115-124). LMpectins can also be extracted with aqueous chelating agents such ashexametaphosphate (e.g. potato pectins) (Voragen et al., 1995, In: Foodpolysaccharides and their applications; Stephen A. M., New York: MarcelDekker Inc, 287-339). The use of the enzyme pectin methyl-esterase (PME)for the production of LM pectins can be an alternative for the chemicalextraction (Christensen, 1986, Pectins. Food Hydrocolloids, 3, 205-230).The conditions and time of the different reactions are varied leading topectins with a different DE, even as low as a DE of zero.

Although, commercial LM pectins are almost exclusively derived from HMpectins, there are natural sources of LM pectin, such as maturesunflower heads (Thakur et al, 1997, Critical Reviews in Food Scienceand Nutrition, 37(1):47-73).

The DE Can be Determined by Commonly Known Methods.

For example, the degree of esterification can be determined usingseveral methods such as titration (Food Chemical Codex, 1981), IRspectrometry (Gnanasambandam & Proctor, 2000, Food Chemistry, 68,327-332; Haas & Jager, 1986, Journal of Food Science, 51(4), 1087-1088;Reintjes et al, 1962, Journal of food sciences, 27, 441-445) and NMRspectrometry (Grasdalen et al, 1988, Carbohydrate Research, 184,183-191). Other methods using HPLC (Chatjigakis et al., 1998,Carbohydrate Polymers, 37, 395-408; Levigne et al., 2002, FoodHydrocolloids, 16(6), 547-550; Voragen et al, 1986, Food Hydrocolloids,1(1), 65-70) and GC-headspace (Huisman et al, 2004, Food Hydrocolloids,18(4), 665-668; Walter et al, 1983, Journal of Food Science, 48(3),1006-1007) analysing the methanol content after saponification of thepectins have been developed. A capillary electrophoresis (CE) method hasbeen developed to determine the DM of the polymers as such (Jiang et al,2005, Food Chemistry, 91, 551-555; Jiang et al, 2001, of Agriculturaland Food Chemistry, 49, 5584-5588; Zhong et al, 1998, CarbohydrateResearch, 308, 1-8; Zhong et al, 1997, Carbohydrate Polymers, 32(1),27-32). An advantage of the CE method is that the GalA content of thesamples is not required to calculate the DM whereas the GalA values haveto be known prior to the DM calculation following GC headspace and HPLCmethods.

Surprisingly it was found that the use of at least one pectin with a DEof less than 65% was able to prevent, reduce and/or treat inflammatorydisorders, diseases, or discomforts.

The pectins according to the present invention are preferably notamidated (no amide groups are in present in the pectin).

Furthermore it is surprising that this effect is achieved with a lowerconcentration of pectins when compared to the use of pectins known fromthe prior art. This leads to advantages in formulation, as lowerconcentrations of the current pectins are easier to formulate in to foodand/or feed products.

Therefore the present invention relates to an esterified pectin or amixture of esterified pectins for the use in the treatment ofimmune-mediated diseases and inflammatory diseases in patients,characterized in that the esterified pectin (or the mixture ofesterified pectins) has a degree of esterification (DE) of less than65%.

The degree of esterification in the context of the present invention ispreferably determined by the HPLC method as described by A. G. J.Voragen, H. A. Schols and W. Pilnik, in the publication titled“Determination of the degree of methylation and acetylation of pectinsby h.p.l.c,”, published in Food Hydrocolloids, volume 1, issue 1, pages65-70, 1986.

Furthermore the present invention relates to a method (M) of preventing,reducing and/or treating inflammatory disorders, diseases, ordiscomforts (lessening inflammatory diseases) by administering topatients esterified pectins (or a mixture of esterified pectins),wherein the pectins have a degree of esterification of less than 65%.

As illustrated by the examples pectins with a higher degree ofesterification (more than the pectin of the present invention; i.e. a DEof 75) are far less effective, and only at higher pectin dosages.

In the context of the present the pectins can be obtained from any knownsources. A list of suitable sources is given above. By using one of theprocesses as described above, the pectins with the correct DE areobtained.

Preferably the DE of the pectin (as a single compound or when used in amixture) is less than 60%, more preferably less than 55%, especiallypreferred less than 50%.

Therefore the present invention also relates to an esterified pectin ora mixture of esterified pectins for the use in the treatment ofimmune-mediated diseases and inflammatory diseases, which is esterifiedpectin or a mixture of esterified pectins, wherein the pectin has a DEof less than 60%.

Therefore the present invention also relates to an esterified pectin ora mixture of esterified pectins for the use in the treatment ofimmune-mediated diseases and inflammatory diseases, which is esterifiedpectin or a mixture of esterified pectins, wherein the esterified pectinor a mixture of esterified pectins has a DE of less than 55%.

Therefore the present invention also relates to an esterified pectin ora mixture of esterified pectins for the use in the treatment ofimmune-mediated diseases and inflammatory diseases, which is esterifiedpectin or a mixture of esterified pectins, wherein the esterified pectinor a mixture of esterified pectins has a DE of less than 50%.

Therefore the present invention also relates to a method (M₁), which ismethod (M), wherein the pectin has a DE of less than 60%.

Therefore the present invention also relates to a method (M₁′), which ismethod (M), wherein the pectin has a DE of less than 55%.

Therefore the present invention also relates to a method (M₁″), which ismethod (M), wherein the pectin has a DE of less than 50%.

Usually the pectin has a DE of at least 1%, preferably of at least 2,more preferably of at least 3%. Therefore there is a range of 1-65%,2-65%, 3-65%, 1-60%, 2-60%, 3-60%, 1-55%, 2-55% and 3-55%.

Commercial pectins can be a mixture of several populations: thedistribution of the substituents can differ in an intramolecular level(within one single pectin polymeric chain) or in an intermolecular level(within one single pectin sample). This holds for all substituents, thusthe sugars as well as the esterifications, and therefore both categoriesare meant with the word substituents' in the following. The substituentscan be distributed completely at random. This random distribution canfollow an even distribution pattern, when the substituents are regularlydistributed over a single pectin polymeric chain, leading to a morehomogenous pectin polymeric chain. If all pectin polymeric chains in asingle pectin sample are of the same homogenous type, also the samplecan be called homogeneous.

However, a single homogenous pectin polymeric chain can be present in acomposition with other homogenous pectin polymeric chains but having adifferent intramolecular (but still homogeneous) distribution of thesubstituents. In this case, the pectin sample should be consideredheterogeneous.

Furthermore it is also possible to modify the esterified pectinaccording to the present invention. One of the possible modifications isamidation. Amidated pectin is a modified form of pectin. In that casesome of the galacturonic acid is converted with ammonia to carboxylicacid amide. This is done according to well-known processes. The presenceof an amide group is typically at the C-6 position of the amidated GalAresidues. If pectin is amidated, the DE is often expressed as the degreeof amidation (i.e. DAM). In that case, the degree of esterification isby definition the amount of amides (in moles) present per 100 moles oftotal galacturonic acids (free GalA and substituted GalA summedtogether).

Other possible modifications of pectins are ethyl or propyl.

Preferably the esterification type of the pectin is either methylationand/or acetylation, more preferably methylation.

Therefore the present invention also relates to an esterified pectin ora mixture of esterified pectins as described above for the use in thetreatment of immune-mediated diseases and inflammatory diseases, whereinthe esterification type of the pectin is either methylation and/oracetylation

Therefore the present invention also relates to an esterified pectin ora mixture of esterified pectins as described above for the use in thetreatment of immune-mediated diseases and inflammatory diseases, whereinthe esterification type of the pectin is methylation.

Therefore the present invention also relates to a method (M₁), which ismethod (M), wherein the esterification type of the pectin is eithermethylation and/or acetylation.

Therefore the present invention also relates to a method (M₁′), which ismethod (M), wherein the esterification type of the pectin ismethylation.

Methods to characterize the different components (i.e. GalA content,neutral sugar content, degree of methyl-esterification, degree ofactetylation, degree of amidation, distribution of thenon-methyl-esterified GalA, molecular weight) of natural, modified aswell as commercial pectins are well described in the PhD thesis ofStéphanie Guillotin (Studies on the intra- and intermoleculardistributions of substituents in commercial pectins. WageningenUniversity, The Netherlands, 2005. ISBN 90-8504-265-8).

As described above the specific pectins are used to combatinginflammatory disease of humans or animals.

The term “patient” means a human or other animal likely to develop orsuffering from inflammatory disease, including avian, bovine, canine,equine, galline, feline, hircine, lapine, murine, musteline, ovine,piscine, porcine and vulpine animals. Preferably, the patient is ahuman, bovine, canine, feline, galline, ovine, porcine or avian.

The specific pectins according to the present invention are used—asdisclosed above—to prevent, reduce and/or treat inflammatory disorders,diseases, or discomforts. This result is obtained by the fact that thespecific pectins according to our invention are surprisingly able tobind TLR2, which is involved in many diseases, in regulation of theintestinal barrier function and in regulation of immune responses.

Toll-like receptors (TLRs) are a class of proteins that play a key rolein the innate immune system. They are single, membrane-spanning,non-catalytic receptors usually expressed in sentinel cells such asmacrophages and dendritic cells, that recognize structurally conservedmolecules derived from microbes. Once these microbes have breachedphysical barriers such as the skin or intestinal tract mucosa, they arerecognized by TLRs, which activate immune cell responses. The TLRsinclude TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10,TLR11, TLR12, and TLR13.

Surprisingly, it was found that only pectins with a low degree ofesterification (i.e. low DE) are able to bind TLR2 in sufficient manner.Specifically, pectins with a low degree of methylation (i.e. low DM) areable to bind TLR2 in sufficient manner.

The interaction of the specific pectins according to our invention withTLRs was tested with cell based assays. In the context of the presentinvention HEK-Blue™ Detection (from InvivoGen) was used. The HEK-Blue™system consists of various specific cell lines and cell culture mediumdeveloped to provide a fast and convenient method to monitor molecularinteractions through so-called SEAP expression (see example 1 fordetails). This cell assay is commercially available. The results ofthese tests are disclosed in all details in the following examples. Itwas shown that low esterified (especially methylated) pectins show asurprising positive effect!

The amount of pectin, which is used can vary, depending on the patient.It must be an amount which shows a sufficient effect.

The pectin, which is ingested by the patient can be in any form. It ispossible to use the pectin as such or in a mixture with otheringredients. When used in a mixture, the amount in this mixture is thendepending on the other ingredients as well as the form of the mixture.

The ingredients which are used are usually chosen with regard to the useof the mixture. The ingredient can serve to improve the properties ofthe mixture or when the mixture is used to be formulated into a finalcomposition to improve the final composition.

The ingredients can serve one or more purposes. It is clear that suchingredients must be food or feed grade (depending on its use).

The pectin can also be part of a food or feed product, whereas the foodor feed product can be in any commonly known and used form.

The amount of pectin, which is used can vary (depending on the patientand/or inflammatory disease targeted). It depends on the body weight.Usually an amount between 0.01 and 5 g pectin with a DE of less than 65%per Kg body weight and per day is desired.

The amount pectin in a specific food or feed product usually variesdependent on the food or feed product. It is also dependent on how mucha consumer/animals eat of this food or feed product. The amount in afood or feed product should be in such an amount that by a usualconsumption of that food or feed product the necessary dosage of pectinis consumed.

The following examples serve to illustrate the invention.

EXAMPLES Example 1 Pectin Inhibition of TLR2-Mediated NFkB Activation

Cell Lines and Cultivation

Cell lines were cultured in DMEM culture media (Lonza, Basel,Switzerland) with 10% decomplemented Fetal Calf serum, 50 U/mlPenicillin (Sigma, St. Louis, Mo., USA), 50 μg/ml Streptomycin (Sigma,St. Louis, Mo., USA) and 100 μg/ml Normocin (InvivoGen, Toulouse,France).

HEK-Blue™ TLR2-CD14 cells (InvivoGen, Toulouse, France) expressing humanTLR2 and SEAP (Soluble Embryonic Alkaline Phosphatase) were used. NFκBand AP1 are stimulated to move to the nucleus in these cell lines whenTLR2 is activated by an agonist. Here, the SEAP gene is under thecontrol of a NFκB/AP-1 responsive promoter. Upon expression, the SEAPgene product is secreted in media and can be quantified using theQuantiblue (InvivoGen, Toulouse, France) solution. The enzyme convertsthe pink color into blue depending on the activity, and this can bemeasured with a spectrophotometer.

1× HEK-Blue™ Selection (InvivoGen, Toulouse, France) was added to thecultivation medium to control only the growth of the HEK-Blue™ TLR2-CD14cells.

Pectin Sources

All pectins are isolated from citrus. Pectin with a DE of 0 was obtainedfrom MP Biomedicals, LLC. The pectins with a DE of 7, 22, 45, 60 and 75were obtained from CP Kelko.

Pectin Inhibition TLR2-1

HEK-Blue™ TLR2-CD14 cells were activated with the TLR2-1 specificagonist Pam3CSK4. Pectins of different DE values (0, 7, 22, 45, 60 and75) were added at 0.5, 1 and 2 mg/ml. As a control HEK-Blue™ TLR2-CD14cells were incubated with only pectin.

HEK-Blue™ TLR2-CD14 cells were seeded at 500,000 cell/ml in a 96 wellplate with 100 μl volume per well. Cells were allowed to grow overnight.The following day, cells were treated with different pectins atdifferent concentrations to study the effect on TLR2. After one hour ofincubation with pectin, Pam3CSK4 was added at a concentration of 100ng/ml. After 24 hrs of incubation at 37° C. with pectin and Pam3CSK4,the expression of the SEAP gene was determined. Supernatant of incubatedcells was mixed with QUANTI-Blue solution in a ratio of 1:10. Presenceof SEAP makes QUANTI-Blue turn blue. The NFκB activation was quantifiedby measuring the color intensity at 650 nm using an ELISA plate readerVersa Max (Molecular devices, Sunnyvale, Calif., USA). The assay wasperformed in 96 well plates with 8 technical repeats. Each experimentwas repeated three times.

NFκB activation through TLR2-1 by Pam3CSK4 with and without pectinControl 0.5 mg/ml 1 mg/ml 2 mg/ml (no Pam3CSK4) Pectin NFκB % NFκB %NFκB % NFκB type activation inhibition activation inhibition activationinhibition activation — 1.89 0 1.89 0 1.89 0 n.a.  0DE 1.22 35.51 1.2732.59 1.23 35.13 0.01  7DE 0.99 47.64 0.77 59.15 0.81 57.05 0.15 22DE1.62 14.00 1.20 36.67 0.57 69.64 0.04 45DE 1.75 7.33 1.32 30.12 0.6466.22 0.03 60DE 1.95 0 1.56 17.36 0.88 53.22 0.05 75DE 1.96 0 1.88 0.520.85 54.93 0.13

Surprisingly, pectins are inhibiting TLR2, as determined by the colorchange of Quanti-blue at 650 nm through the level of SEAP activity afterNFκB activation of the SEAP gene expression. The inhibition was highestwith pectins with a low DE, but the inhibition was found with all DEvalues depending of the concentration of the respective pectin. Theinhibiting effect of pectins with higher DE values (45, 60 and 75) wasclearly concentration dependent, suggesting that at higherconcentrations the absolute number of intra- or intermolecular regionswith a local DE<65% (due to the possible irregular or heterogeneousdistribution of esterified GalA residues throughout the pectin moleculesand/or sample) becomes higher and therefore the inhibition ofTLR2—through those regions with DE lower than 65—increases.

The viability of the HEK-Blue™ TLR2 CD14 was not affected by theadditions of pectin (tested using WST-1 reagent).

Example 2 Pectin Binds to TLR2

Construction of the TLR2Ectodomain-HA Expression Plasmid

RNA was extracted from HEK-Blue™ hTLR2-CD14 cells using the RNeasy®PlusMini kit (Qiagen, Venlo, Netherlands). cDNA was synthesized usingOligodT primers (Life technologies, Carlsbad, Calif., USA), dNTP mix(Life technologies, Carlsbad, Calif., USA) and Superscript™III ReverseTranscriptase (Life technologies, Carlsbad, Calif., USA) according tothe suppliers' manuals. TLR2 from codon1 to codon586 was synthesizedusing cDNA from HEK-Blue™ hTLR2-CD14 using the forward primer5′-GCGCACCGGTATGCCACATACTTTGTGGATGG-3′ (SEQ ID NO: 1), the reverseprimer 5′-GCGCGGATCCGTGACATTCCGACACCGAGAG-3′ (SEQ ID NO: 2) and Pfu DNApolymerase (Thermo scientific, Waltham, Mass. USA). Primers were flankedby a GC doublet at the 5′ end for restriction enzyme recognition. AgeIand BamHI restriction sites were included in the forward and reverseprimer, respectively. The PCR product was digested with AgeI and BamHIrestriction enzymes (Thermo scientific, Waltham, Mass. USA) and theplasmid pSELECT-CHA-blasti (InvivoGen, Toulouse, France) to createsticky ends. The PCR-amplified TLR2 ectodomain fragment and linearplasmid were ligated using T4 DNA Ligase (Thermo scientific, Waltham,Mass. USA). The ligated plasmid was used to transform One Shot TOP10Chemically Competent E. coli (Life technologies, Carlsbad, Calif., USA).Transformed E. coli cells were selected using Blasticidin agar media(InvivoGen, Toulouse, France). Obtained colonies were screened forcorrect orientation of the gene in the plasmid. Selected correctcolonies were grown in blasticidin liquid media (InvivoGen, Toulouse,France) and the plasmid was isolated using the Qiagen Midi prep kit.Plasmid was then sequenced for selection of non-mutated clones(Baseclear, Leiden, Netherlands).

Transfection of HEK293T With the TLR2Ectodomain-HA Expression Fragment

HEK293T cells were seeded at 500,000 cells/ml in 12 well culture platesand incubated overnight. The following day, transfection was performedby using Lipofectamine LTX® (Life technologies, Carlsbad, Calif., USA).The sequence verified plasmid was linearized with the restriction enzymeNotI (Fast digest, Thermo Scientific, Waltham, Mass. USA). Purified, 1μg linear plasmid was diluted in low serum media Opti-MEM® (Lifetechnologies, Carlsbad, Calif., USA) and mixed with 3.5 μl ofLipofectamine LTX® (Life technologies, Carlsbad, Calif., USA). Thistransfection mix was incubated for 30 min at room temperature and thenadded to the previously seeded cells in the culture media. Cells wereincubated with transfection medium mix for 24 hrs and transfected cellswere selected using blasticidin in DMEM culture media (Lonza, Basel,Switzerland) with 10% decomplemented fetal calf serum, 50 U/mlPenicillin (Sigma, St. Louis, Mo., USA), 50 μg/ml Streptomycin (Sigma,St. Louis, Mo., USA) and 100 μg/ml Normocin (InvivoGen, Toulouse,France). Single cell clones were isolated to form the HEK293TTLR2ectodomain-HA cell line.

Cell Cultivation

Cell lines were cultured in DMEM culture media (Lonza, Basel,Switzerland) with 10% decomplemented Fetal Calf serum, 50 U/mlPenicillin (Sigma, St. Louis, Mo., USA), 50 μg/ml Streptomycin (Sigma,St. Louis, Mo., USA), 100 μg/ml Normocin (InvivoGen, Toulouse, France)and 50 μg/ml blasticidin (InvivoGen, Toulouse, France).

Protein Immunoprecipitation

Overnight grown HEK293T TLR2ectodomain-HA cells were lysed using 1× RIPAlysis buffer (Merck Millipore, Billerica, Mass., USA) in the presence ofa protease inhibitor cocktail consisting of AEB SF(4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride), Aprotinin,Bestatin, E-64, EDTA and Leupeptin (Sigma, St. Louis, Mo., USA) at 4° C.for 10 min followed by two times sonification for 5 seconds at 0% power.Supernatant was isolated after centrifugation at 14000 g for 10 min.TLR2ectodomain-HA tagged protein was immunoprecipitated using Pierce®Anti-HA Agarose (Thermo scientific, Waltham, Mass. USA) in a microcentrifuge tube. Protein was competitively eluted using HA syntheticpeptide (Thermo scientific, Waltham, Mass. USA) by incubating two timesfor 15 min with a single bed volume of HA synthetic peptide at 30 C.Isolated protein was desalted and HA peptide was removed using Zeba SpinDesalting Columns and Devices, 40K MWCO (Thermo scientific, Waltham,Mass. USA). Isolated and desalted protein was quantified using Thermoscientific BCA protein assay kit.

ELISA for Binding of TLR2 and Pectin

The ELISA buffer was composed of 1 mM CaCl2 and 150 mM NaCl in 0.05MTris buffer at pH 8.2. The buffer was used for washing as well asdiluent for antibodies and pectin. The blocking buffer was made bydissolving 3% milk powder (FrieslandCampina, Amersfoort, TheNetherlands) in ELISA buffer. For antibody solutions, a 1:2 dilution ofblocking buffer with ELISA buffer was used for dissolving antibodies.ELISA plates (Corning, Tewksbury, Mass., USA) were treated with 50 μl of50 μg/ml of Poly-L lysine for 1 hour at 37 C. Wells were washed oncewith 400 μl ELISA buffer. Pectins (0, 7, 22, 45 60 and 75DE) weredissolved in ELISA buffer at 1 mg/ml concentration and 50 μl was addedto each well. The plates were incubated for 4 hours at 37 C to allow forbinding of the pectin. Each well was then washed with 400 μl ELISAbuffer and blocked overnight with 100 μl of blocking buffer at 4 C.After the blocking step, the ELISA plate was washed once with ELISAbuffer. TLR2 ectodomain-HA fusion protein was applied to the pectincoated wells at 0.33 μg, 1 μg, 3 μg and 9 μg concentration per well. TheELISA plates were then incubated at 37 C for 3 hrs. The HA syntheticpeptide was used as a negative control at 0.33 μg, 1 μg, 3 μg and 9 μgconcentration per well, same as with the TLR2 ectodomaian-HA fusionprotein. Pectin binding antibodies LM19 (specific for DE 0 and 7) andLM20 (specific for DE 22, 45, 60 and 75) from Plantprobes (Leeds, UK)were used as positive control for pectin binding at 1:100 dilutions.Afterwards, the wells were washed with 400 μl of ELISA buffer for 5times and incubated with 50 μl primary detection antibody for the HA tag(Cell Signaling, Danvers, Mass., USA) in a 1:200 dilution. The primaryantibody was incubated for 2 hours at 37 C. After primary antibodyincubation, the plates were washed again 5 times with ELISA buffer.After washing, 50 μl of Biotin tagged secondary antibody (SouthernBiotech, Birmingham, Ala., USA) was applied to each well at a 1:500dilution. The Biotin tagged antibody was incubated for 1 hr at 37 C.This step was followed by 5 washings with 400 μl of ELISA buffer.Streptavidin-HRP (Dako, Heverlee, Belgium) (100 μl) was added to eachwell at a 1:1000 dilution. After incubation at 37 C for 1 hr, plateswere washed 7 times with 400 μl of ELISA buffer. Finally, for detection,100 μl of TMB substrate (Cell Signaling, Danvers, Mass., USA) wasapplied to each well and incubated at 37 C for 30 min. The reactionswere stopped by adding 100 μl of stop solution (Cell Signaling, Danvers,Mass., USA). The ELISA plate was read at 420 nm in Plate reader VersaMax (Molecular devices, Sunnyvale, Calif., USA). For clarity sake thevalues of the negative control are subtracted from the values obtainedwith TLR2 ectodomain-HA.

TLR2 ectodomain binding to pectin (a.u.) 0.33 μg 1 μg 3 μg 9 μg Pectintype protein protein protein protein 0DE 0.03 0.15 0.45 1.43 7DE 0.450.98 2.41 2.34 22DE 0.03 0.16 0.49 0.81 45DE 0.13 0.25 0.58 0.26 60DE0.07 0.09 0.16 0.07 75DE 0.00 0.03 0.06 0.03

Surprisingly, pectins with low DE are directly binding to the TLR2ectodomain, as indicated by the dose-dependent values obtained in theELISA test.

Example 3 Pectin Inhibits Only the Pro-Inflammatory Pathway of TLR2 andNot Its Regulatory Pathway

Cell Line

HEK-Blue™ Null1 (InvivoGen, Toulouse, France) was seeded at 500,000cells/ml in 12 well culture plates and incubated overnight. Thefollowing day, transfection was performed by using Lipofectamine LTX®(Life technologies, Carlsbad, Calif., USA). Plasmid pUNO3-hTLR2(InvivoGen, Toulouse, France) was linearized with NotI (Thermoscientific, Waltham, Mass. USA). One μg of purified DNA was diluted inlow serum media Opti-MEM® (Life technologies, Carlsbad, Calif., USA) andmixed with 3.5 μl of Lipofectamine LTX® (Life technologies, Carlsbad,Calif., USA). This transfection mix was incubated for 30 min at roomtemperature and then added to the previously seeded cells in the culturemedia. Cells were incubated with transfection medium mix for 24 hrs andtransfected cells were selected using Zeocin (100 μg/ml) and HygromycinB (150 μg/ml) in DMEM culture media (Lonza, Basel, Switzerland) with 10%decomplemented fetal calf serum, 50 U/ml Penicillin (Sigma, St. Louis,Mo., USA), 50 μg/ml Streptomycin (Sigma, St. Louis, Mo., USA) and 100μg/ml Normocin (InvivoGen, Toulouse, France). Single cell clones wereisolated. The thus obtained HEK-Blue™ Null1 TLR2 cell line expressesonly TLR2 and not CD14.

TLR2 Activation and Inhibition Assay

HEK-Blue™ Null1 TLR2 cells were seeded at 500,000 cell/ml in a 96 wellplate with 100 μl volume per well. Cells were allowed to grow overnight.The following day, cells were treated with different pectins to studythe effect on TLR2. After one hour of incubation with pectin, the TLR2-6specific agonist FSL-1 was added at a concentration of 100 ng/ml. After24 hrs of incubation at 37 C with pectin and FSL-1, the expression ofthe SEAP gene was determined. Supernatant of incubated cells was mixedwith QUANTI-Blue solution in a ratio of 1:10. Presence of SEAP makesQUANTI-Blue turn blue. The NFκB activation was quantified by measuringcolorimetric readings at 650 nm using ELISA plate reader Versa Max(Molecular devices, Sunnyvale, Calif., USA). The assay was performed in96 well plates with 10 technical repeats. Each experiment was repeatedthree times.

NFκB activation through TLR2-6 by FSL-1 with and without pectin 0.5mg/ml 1 mg/ml 2 mg/ml Control (no FSL-1) Pectin NFκB NFκB NFκB NFκB typeactivation activation activation activation — 1.14 1.14 1.14 n.a. 0DE1.17 1.18 1.15 0 7DE 1.05 1.05 1.08 0 22DE 1.10 1.12 1.02 0 45DE 1.131.11 1.03 0 60DE 1.13 1.17 1.17 0 75DE 1.13 1.13 1.20 0

The data shows that pectin does not inhibit the TLR2-6 signaling pathwayas the stimulus in HEK-Blue™ Null1 TLR2 cells through FSL-1 is notchanged by the presence of pectins, whereas the stimulation by Pam3CSK4was clearly showing that pectin inhibits the TLR2-1 signaling response(example 1).

Example 4 Pectin in the Diet of Piglets Leads to a Reduced IntestinalPermeability of the Tight Junctions

Piglet Feeding Trial Set-Up

The experimental farm for young piglets is located in Flanders (Belgium)and consists of 8 batteries, each containing 4 pens. The piglets understudy are hybrids of Topigs Piétrains and are weaned at 21 days. Thepiglets are weighed individually at weaning and 2 and 4 weeks afterweaning. Feed intake is registered per pen of 4 piglets at the momentsof weighing. At arrival the piglets are earmarked with a newSanitel-number. During the trial, a veterinarian and a Felasa Dcertified person supervise the performed piglet experiment according tothe international guidelines described in law EC/86/609.

Each pen (1.5 m×1.5 m) contains 4 piglets at the start of the trial. Foreach pen, one feeder (ad libitum) is installed for meal or pellets. Onedrinking nipple is installed per pen. The temperature at start is at28±2° C. until 10 days after weaning. Afterwards, temperature isdecreased to 25±2° C.

Commercial non-medicated diets are given. Non-medicated means that thepiglet doesn't receive any therapeutic antibiotics before and during thetrial. The diets are given in the form of meal. All feed were analysedfor their nutritional content.

Four treatments were applied (diets A, B, C, D) on 7 replicates with 4piglets per group. At the start of the trial, the piglets (around 7 kgbody weight) are allocated to the different pens by weight. Thisallocation is made in order to have an equal average weight and an equalstandard deviation around the average weight for each treatment and pen.For microbiological counting's and for taking biopsis, piglets receivean overdose of barbiturates (Nembutal) followed by sacrification.Afterwards, a section is performed on the piglets. Samples for microbialcounts are immediately processed, while samples taken for histochemicalexperiments were fixed for later analysis. During the whole trial periodthe piglets are fed ad libitum, except for the period of microbiologicalcountings. At that moment, three days before the microbiologicalcountings are performed, the piglets are fed restricted. Piglets receivethree times a day an amount of feed, which is carefully weighed andnoted. The feed is given at 8.00, 13.00 and 18.00. When necessary, thesick piglets were treated individually (by injection). The followingparameters were taken into account. (i) individual growth data, (ii)feed intake data per pen (corrected for eventual losses), (iii) feedconversion ratio during weaning, starter and whole trial period, (iv)fecal score and clinical score, (v) tight Junctions, (vi) microbialanalysis, (vii) histochemical analysis.

Diets

Feed Compositions (in g/kg):

Ingredient Feed A Feed B Feed C Feed D Corn 171.13 169.13 169.13 169.13Grains (wheat and barley) 491.83 491.83 491.83 491.83 Protein sources(soy, potato) 227.72 227.72 227.72 227.72 Milk derivatives (whey) 52.6552.65 52.65 52.65 Soy bean oil 14.19 14.19 14.19 14.19 Amino acids 10.8010.80 10.80 10.80 Minerals & trace minerals 10.24 10.24 10.24 10.24Limestone 10.63 10.63 10.63 10.63 Enzyme* 0.64 0.64 0.64 0.64 Premix**10.17 10.17 10.17 10.17 Pectin DE33 — 2.00 — — Pectin DE55 — — 2.00 —Pectin soybean meal — — — 2.00 *Xylanase/beta-glucanase and phytasecocktail (BASF) **Premix includes aroma's, extra trace minerals,vitamins (Vitamex N.V.)

Pectin Sources

Pectins with a DE 33 and 55 were isolated from citrus and obtained fromHerbstreith & Fox (Neuenbürg/Württingen, Germany). The Soy Bean Meal(SBM) was from South-American origin (mixture from Argentina, Brasiland/or Paraguay) and processed to extract the residing pectins by mixingthe SBM at 33% dry matter with tapwater and autoclaving for 30 mins at120 C. After cooling the obtained material was freeze dried and milled,and used as such in the diet.

Mannitol-Lactulose Test

The permeability of the ileum was quantified by performing amannitol-lactulose test in the animals. Lactulose cannot pass an integersmall bowel which is considered to be positive as there is a lowerchance on systemic infections and immune issues.

Mannitol is a metabolically inert monosaccharide, which is passivelyabsorbed through the intestinal mucosa. Any absorbed mannitol is fullyexcreted in the urine within a couple of hours. Mannitol wasadministered to the piglets through a stomach-pump at 0.3 g mannitol/kgbody weight (4 hours before dissection). Lactulose is a metabolicallyinert disaccharide, which normally is not absorbed unless the mucosalbarrier is compromised. Any absorbed lactulose is fully excreted in theurine within 6 hours. Lactulose was administered to the piglets througha stomach-pump at 0.75 g lactulose/kg body weight (4 hours beforedissection)

During dissection piglet urine was then collected. In piglets with ahealthy intestine, the mean absorption of lactulose is less than 1% ofthe administered dose. A recovery of >1% lactulose in the urineindicates a disaccharide hyperpermeability.

In piglets with a healthy intestine, the mean absorption of mannitolis >14% of the administered dose.

A recovery of <14% mannitol in the urine indicates a carbohydratemalabsorption.

A lower lactulose/mannitol ratio (L/M ratio) indicates a positive effectof a diet.

Permeability test of the gastrointestinal tract (average of threeindividual pigs per diet) Diet L/M ratio SD A 0.55 0.90 B 0.019 0.032 C0.024 0.042 D 0.15 0.27

As shown all pectins, and especially the pectins with DE 33 and 55pectin, lead to a reduced L/M ratio and thus have a positive effect inthe small intestine of the young pigs

Example 5 Pectin in Diets Lead to an Increase of the Villus to CryptRatio

The piglet feeding trial set-up was as described in example 4.

Sample Preparation for Histology

Sampling: Take a sample of the duodenum and/or ileum and rinse withphysiological water (0.9% NaCl). Store in 20 ml formalin buffer (1 mlformaldehyde (37%)/liter). 4.5 g NaH₂PO₄+10.4 g Na₂HPO₄)

Bedding: Poor the paraffin solution in the recipient (not fully full)and place the intestinal sample vertical in the recipient. Let solidifyat 4° C. and fill the recipient completely. Let solidify at −3° C.

Biopt: Clean scalpel with 10% xylene and let dry. Transfer coupe withbrush and needle to 50% alcohol. Cut in pieces and transfer to distilledwater (65° C.) with brush and microscope slide. Place sample on themicroscope slide and incubate one night at 60° C.

Haematoxiline-eosine (H&E) staining steps at ambient temperature:

-   -   1). Deparaffinize        -   Wash 3 times with 10% xylene (for 5 minutes)        -   Wash 2 times with 100% ethanol (for 3 minutes)        -   Wash once with ethanol 90% (for 3 minutes)        -   Wash once with ethanol 70% (for 3 minutes)        -   Wash once with water (for 3 minutes)        -   Wash once with water (for 3 minutes)    -   2). staining        -   Incubate in Mayers haematoxiline (for 6 minutes)        -   Wash once with water (for 5 minutes)    -   3). counter-staining        -   Incubate in 10% eosine (for 5 minutes)        -   Wash 10 times with water (each for 30 seconds)    -   4). Dehydrate        -   Wash 10 times with ethanol 90% (each for 30 seconds)        -   Wash 10 times with ethanol 70% (each for 30 seconds)        -   Wash 2 times with ethanol 100% (for 5 minutes)        -   Wash 3 times with 10% xylene (for 5 minutes)

Quantification of the Villi Length and Crypt Depth

Above embedded histological samples are analysed by means of an Olympusmicroscope, and villi length (mm) and crypt (depth) are measured.

Histological morphology of the gastrointestinal tract (average of 3 pigsper diet) Diet SI Crypt (mm) Villus (mm) V/C ratio A Duodenum 23.5 ± 3.336.6 ± 7.3 1.6 ± 0.3 Ileum 15.0 ± 0.8 22.0 ± 3.0 1.5 ± 0.2 B Duodenum23.7 ± 4.7 40.5 ± 3.5 1.8 ± 0.3 Ileum 15.2 ± 0.5 25.1 ± 0.7 1.7 ± 0.1 CDuodenum 23.4 ± 1.9 42.5 ± 8.1 1.8 ± 0.4 Ileum 15.3 ± 1.2 26.7 ± 4.1 1.8± 0.1 D Duodenum 23.8 ± 2.7 37.0 ± 3.9 1.6 ± 0.1 Ileum 15.2 ± 2.6 21.5 ±1.7 1.5 ± 0.4

Longer villi imply a higher absorption capacity while longer cryptsimply the opposite. Thus a higher villus to crypt ratio is considered tobe a positive effect of a diet, while a lower villus to crypt ratio isconsidered to be a negative drawback for using the respective diet. Asshown, especially the diets with pectin DE 33 and 55 have an enhancedvilli-to-crypt ratio in both the duodenum and ileum, which demonstratethe potential of both pectins to improve gastrointestinal health.

Example 6 Pectin Acts as Anti-Inflammatory Agent in Doxorubicin InducedMucositis

Mucositis, also referred to as mucosal barrier injury, is one of themost severe side effects of radiotherapy and chemotherapy treatment.Both inflammation and apoptosis of the mucosal barrier result in itsdiscontinuity, thereby promoting bacterial translocation. Five phasesare important in the pathophysiology of mucositis: (1) the formation ofreactive oxygen species leading to the activation of nuclear factorkappa B (NFkB) during the initiation phase, (2) the induction ofmessenger molecules such as tumor necrosis factor alpha (TNFa),resulting in treatment-related tissue inflammation and apoptosis duringthe upregulation/message generation phase, (3) the amplification ofmessenger molecules in the amplification/signaling phase, leading tomore inflammation and apoptosis, (4) discontinuity of the epithelialbarrier resulting from apoptosis during the ulcerative phase, therebypromoting bacterial translocation, and (5) a spontaneous healing phase,characterized by cell proliferation (Sonis, 2004, Semin Oncol Nurs.20(1):11-5); Van Vliet et al, 2010, PLoS Pathog. 6(5):e1000879).

Mice

C57B1L/6 female mice (7-10 weeks old) were purchased from Janvierlaboratories, France. The experimental use of animals was approved bythe Animal Ethical Committee of the University of Groningen. All themice were acclimatized for 2.5 weeks prior to start of the experiment.Mucositis was induced by administration of doxorubicin (Sigma, St.Louis, Mo., USA).

DietsMice were supplied with ad-libitum RMH-B diet (AB diets, Woerden,The Netherlands). The ingredients of diet specified by supplier arewheat, meat meal (80% sterilized), yellow dent corn, whole oats, wheatmiddlings, alfalfa, soya oil, dried yeast, dicalcium phosphate, calciumcarbonate, NaCl, dl-methionine, vitamins and trace elements. Mice weresupplied with drinking water from tap and the water bottles were changedonce a week.

Pectin Sources

The pectin with a DE of 7 was obtained from CP Kelko.

Induction of Mucositis and Readouts

Doxorubicin was dissolved in sterile 0.9% sodium chloride and stored inaliquots at 4 C. Pectin (7DM) was dissolved in sterile water andadministered by gavage to mice for 10 or 11 days, twice a day at 3mg/day. On day 8, doxorubicin was injected intra-peritoneal at 10 mg/kgconcentration. Mice were sacrificed on day 10 (48 hour doxorubicin) orday 11 (72 hour doxorubicin). Animals receiving water by gavage servedas controls. After the collection of tissue samples, mice weresacrificed by cervical dislocation.

TLR2 Blocking in Mice

TLR2 blocking antibody, clone T2.5 (InvivoGen, Toulouse, France) wasadministered IP at 10 mg/kg one hour prior to doxorubicin treatment.

Neutrophil Count

Peritoneal lavage was collected with 2 ml PBS to collect peritonealneutrophil influx. The total number of living cells in the peritoneallavage was counted using a Z™ Series coulter Counter® (Beckman Coulter,Brea, Calif., USA). The lavage was diluted at 500,000 cells/ml and 100μl of cell solution was applied for cytospin preparation. The cytospinslides were stained with Giemsa stain (Merck Millipore, Billerica,Mass., USA) for 1 hour at room temperature. The stained slides werescanned in a Hamamatsu slide scanner (Hamamatsu photonics, Japan) andneutrophils were counted using morphological features in 250 cells. Thetotal number of neutrophils were calculated using the total cell countin peritoneum and the neutrophoil counts from cytospin preparations.

Effect of 48 hour doxorubicin treatment on neutrophil count Relativeincrease in Mice groups Total number of neutrophils neutrophils Control(n = 6) 3.97E+06 ± 2.39E+06 1.0 Pectin DE7 (n = 6) 2.97E+06 ± 1.29E+060.7 Doxorubicin (n = 6) 2.23E+08 ± 6.74E+07 56.2 Pectin DE7 +Doxorubicin 5.44E+07 ± 2.30E+07 13.7 (n = 5) TLR2 blocking + Doxorubicin7.04E+07 ± 7.30E+07 17.7 (n = 6) TLR2 blocking (n = 5) 8.96E+06 ±7.90E+06 2.3

As can be seen from the data, doxorubicin induced mucositis increasesthe neutrophil count by 56 times. Blocking TLR2 either by the TLR2blocking antibody clone T2.5 or pectin DE7 significantly reduced theneutrophil count, demonstrating that pectin acts as an anti-inflammatoryagent.

Example 7 Pectin Has a Positive Effect on Health Beneficial Microbes

Digesta Collection

The pig fecal samples were collected on day 14 and 28 during theexperimental diet feeding (example 4). After the fecal collectionperiod, animals were anesthetized and euthanized. Digesta samples werecollected from terminal ileum, proximal colon, mid colon and distalcolon. Part of each digesta was stored in 1.5 mL Eppendorf tubes foranalysis of microbiota composition and SCFA. These tubes wereimmediately frozen in liquid nitrogen and stored at −80° C. Theremaining amount of digesta was immediately stored at −20° C. untilfurther analysis.

DNA Extraction and Microbiota Analysis

Microbial DNA was extracted from 250 mg of digesta by using a fecal DNAextraction protocol (Salonen A, Nikkilä J, Jalanka-Tuovinen J, ImmonenO, Rajilié-Stojanovié M, Kekkonen R A, Palva A & de Vos W M. 2010.Comparative analysis of fecal DNA extraction methods with phylogeneticmicroarray: Effective recovery of bacterial and archaeal DNA usingmechanical cell lysis. Journal of Microbiological Methods, 81: 127-134).The DNA is isolated by sequential precipitations and finally purified byusing the QIAamp DNA Stool Mini Kit columns (Qiagen, Hilden, Germany)according to the manufacturer's recommendations. 16S rRNA gene wasamplified and sequenced in paired-end mode by using the MiSeq platform(Illumina).

Sequence Analysis

Raw Illumina fastq files were demultiplexed, quality-filtered andanalyed using QIIME 1.9.0.

Relative abundance of Prevotella species in the microbiota compostion ofexperimental fed pigs Relative Relative Diet Pectin Fecal sampleabundance* increase** A control Terminal ileum 0 — A control Proximalcolon 0.05 1   A control Mid colon 0.11 1   A control Distal colon 0.091   B DE 33 Terminal ileum 0 — B DE 33 Proximal colon 0.51 9.99 B DE 33Mid colon 0.58 5.49 B DE 33 Distal colon 0.55 6.16 C DE 55 Terminalileum 0 — C DE 55 Proximal colon 0.49 9.55 C DE 55 Mid colon 0.41 3.90 CDE 55 Distal colon 0.45 5.07 D Soy Bean Terminal ileum 0 — D Soy BeanProximal colon 0.21 4.04 D Soy Bean Mid colon 0.22 2.09 D Soy BeanDistal colon 0.30 3.36 *The relative abundance is the % of 16S rRNA dataof Prevotella in the total data set obtained through Illumina sequencing**The relative increase is the fold increase in % of Prevotella 16S rRNAin the total data set. i.e. the relative abundance determined for aspecific pectin fed sample divided by the control sample.

Surprisingly, addition of pectin to the diet leads to an increasedprevalence of Prevotella species in the gut (which is an indicator ofhealth, see Wu G D, Chen J, Hoffmann C, Bittinger K, Chen Y Y, KeilbaughS A, Bewtra M, Knights D, Walters W A, Knight R, Sinha R, Gilroy E,Gupta K, Baldassano R, Nessel L, Li H, Bushman F D & Lewis J D. 2011.Linking long-term dietary patterns with gut microbial enterotypes.Science 334:105-108).

All these examples clearly and surprisingly show that the specificpectins show improvements, which are significant.

The invention claimed is:
 1. A method to ameliorate or to lessenimmune-mediated diseases or inflammatory diseases caused by activationof TLR2, wherein the method comprises administering by ingestion to apatient in need thereof a mixture of esterified pectins comprising aneffective amount between 0.01 and 5 g per kg body weight of the patientof a low esterified pectin having a degree of esterification (DE) ofless than 55% obtained from de-esterification of methylated pectin,wherein the patient is an animal selected from the group consisting ofhuman, avian, bovine, canine, equine, galline, feline, hircine, lapine,musteline, ovine, piscine, porcine and vulpine animals.
 2. The methodaccording to claim 1, wherein the DE of the low esterified pectin isless than 50%.
 3. The method according to claim 1, wherein the DE of thelow esterified pectin is at least 1%.
 4. The method according to claim1, wherein the DE of the low esterified pectin is at least 2%.
 5. Themethod according to claim 1, wherein the DE of the low esterified pectinis at least 3%.
 6. The method according to claim 1, wherein theadministering is practiced by feeding a food or feed formulation whichcomprises the mixture of esterified pectins to the patient.
 7. Themethod according to claim 1, wherein the DE of the low esterified pectinis less than 33%.
 8. The method according to claim 1, wherein the DE ofthe low esterified pectin is less than 22%.
 9. The method according toclaim 7, wherein the DE of the low esterified pectin is at least 1%. 10.The method according to claim 8, wherein the DE of the low esterifiedpectin is at least 1%.